Spatio-temporal analysis of prostate tumors in situ suggests pre-existence of treatment-resistant clones

The molecular mechanisms underlying lethal castration-resistant prostate cancer remain poorly understood, with intratumoral heterogeneity a likely contributing factor. To examine the temporal aspects of resistance, we analyze tumor heterogeneity in needle biopsies collected before and after treatment with androgen deprivation therapy. By doing so, we are able to couple clinical responsiveness and morphological information such as Gleason score to transcriptome-wide data. Our data-driven analysis of transcriptomes identifies several distinct intratumoral cell populations, characterized by their unique gene expression profiles. Certain cell populations present before treatment exhibit gene expression profiles that match those of resistant tumor cell clusters, present after treatment. We confirm that these clusters are resistant by the localization of active androgen receptors to the nuclei in cancer cells post-treatment. Our data also demonstrates that most stromal cells adjacent to resistant clusters do not express the androgen receptor, and we identify differentially expressed genes for these cells. Altogether, this study shows the potential to increase the power in predicting resistant tumors.


Supplementary
1: Spearman's rank correlation coefficient between assessed intensity of nuclear AR and repair proteins Ku70 and P-DNA-PKcs. Immunostaining from the fluorescent-labeled AR to the DNA-repair proteins from all individual epithelial cell nuclei across the tissue section was compared, demonstrating that high levels of AR predicts high levels of repair proteins.

Gene Cancer correlation
DHCR24, TRPM8, IF16 DHCR24 is involved in cholesterol biosynthesis and regulated by the AR, and participates in the conversion of adrenal androgen into its more potent forms; testosterone and dihydrotestosterone. Blocking these pathways might help to reduce treatment resistance. The androgen regulated TRPM8, might play an important role in proliferation and apoptosis, and has further been associated with various cancers. IFI6 is an antiapoptotic protein that promotes metastasis in breast cancer.

CD74
The membrane receptor CD74 is upregulated in the non-responsive areas both in patient 1 and 2, and promotes an increased proliferation, migration and metastatic potential in several cancers. It has a role in NF-κB activation, leading to an immunosuppressive environment. It also plays a role in chemokine production of the potent CCL2, enabling attraction of myeloid-derived suppressor cells and TAMs to the tumor. CD74 interacts with the proinflammatory cytokine macrophage migration inhibitory factor (MIF) and has shown to induce EMT.

TIMP1
A secreted glycoprotein that has been linked to promotion of tumor progression by inhibition of apoptosis and stimulation of prostate cancer cell growth. Elevated levels of TIMP1 levels in plasma predict worse survival outcome in metastatic CRPC patients.

IGFBP7, MGP
The tumor stroma marker IGFBP7 regulates the insulin pathway and has been shown to be elevated in invasive prostate neoplasms. MGP expression is known to be upregulated in CAFs in PCa.
Matrix Gla-protein (MGP) belongs to the family of matricellular proteins (MCP) and induces changes in the extra cellular matrix (ECM) crosslinking and is linked to migration.

A2M
Enzymatic activity of PSA-A2M is present in the serum of men with advanced prostate cancer, which in turn affect a range of growth factors such as IL-6, TGF-beta, PDGF, and FGF, leading to tumor microenvironmental changes.  with given gene expression profiles (factors) and gene activity maps, and AR staining to determine spatial localization of nuclear AR. b, Annotation of the combined data per patient. c, Differential gene expression analysis was performed on spot level to compare responding to non-responding factors and sAR(+) areas to sAR(-) areas, and presented as heatmaps. Abbreviations: AR; androgen receptor, ADT; androgen deprivation therapy, sAR(+); stromal nuclear AR positive, sAR(-); stromal nuclear AR negative, ST; Spatial Transcriptomics, STD; Spatial Transcriptome Decomposition Supplementary Figure 5: Spatial Transcriptomics quality control assay. a, Schematic of the Spatial Transcriptomics quality control assay. Tissue sections are placed onto glass slides with microarrays uniformly coated with poly-T capturing oligonucleotides. Permeabilization allows mRNAs to be captured onto an array followed by reverse transcription with incorporation of Cy3-labeled nucleotides. Removal of the tissue section allows for measuring the cDNA footprint. b, Different permeabilization times and pepsin concentrations were tested on two consecutive tissue sections (only singlets shown) to minimize risk for including non-biological-relevant Cy3-aggregations. All pepsin concentrations had 0.1 M HCl. c, cDNA footprint signal was estimated by taking intensity subtracted with background on three epithelial and three stroma cell areas per duplicate using GenePix Pro (87/91 in Brightness/Contrast). This gave six values per treatment when averaging the duplicates. Background on each well was determined by taking the average of three intensity values on the background area. Scale bars 1 mm. d, Boxplot showing net intensities (n=144 tissue areas). The boxes show the mean and quartiles of the dataset while the whiskers extend to 1.5 of the interquartile range, points beyond the whiskers are considered to be outliers. The highest values for both epithelial and stromal areas is seen at 11 min with 0.013% and these parameters were chosen for further experiments. Source data is provided as Source Data file.    IGFBP7  A2M  VIM  B2M  MGP  TAGLN  LUM  CST3  IGFBP4  DCN   IGHA1  IGKC  IGHA2  JCHAIN  TAGLN  MYL9  CST3  MYH11  EEF2  KLK2   IGKC  IGHG3  IGHG2  IGHG1  IGHG4  IGHGP  IGHA1  TAGLN  B2M TGM2  KLK3  KLK2  TMSB10  CD74  HLA-DRA  CALR   IGLC3  IGLC2  IGHA1  IGHA2  TAGLN  JCHAIN  IGHM  MYL9  MGP  IGLC6 IGKC  IGHG4  IGLC3  IGHG3  IGHA1  IGHG1  IGHG2  IGHGP  IGLC2  IGHA2   0 KLK3  ACPP  KLK2  TFF3  AZGP1  TRPM4  H2AFJ  TSPAN1  MSMB  FASN   AZGP1  KLK2  KLK3  SNHG25  NDRG1  EEF2  RACK1  CFD  DHCR24  IGFBP7   0     . Circles indicate the ratio of epithelial and stroma spots as representative of the active transcriptomic factors. Each transcriptomic factor was mapped to an annotation and to the AR activity in the nucleus. 'B' corresponds to presence before treatment and 'B/A' to presence before and after treatment. Source data is provided as Source Data file. Abbreviations: AR; androgen receptor, ADT; androgen deprivation therapy, F; factor, PCa; prostate cancer AR-epith AR-epith  Fig. 17: Correlation of cancer factors to AR activity. a, Examples of spatial distribution of spots, pre-and post-ADT, representative of responding and non-responding factors pre-and post-ADT. As expected, nuclear AR of epithelial cells pre-ADT was detected irrespectively of responsiveness, while nuclei AR of epithelial cells post-ADT was only seen in non-responsive tissue areas. The core needle biopsies of patient 2 were originally connected on the bottom. Scale bars on HE images corresponds to 1mm and bars on immunostained close ups corresponds to 500 #m. b, Zoom-in immunostained image showing that the AR of epithelial cells is nuclear. Scale bar corresponds to 10 #m. Scale bar, 1 mm. b, The signal of the AR transformed to a heatmap with the same resolution as the ST data. c, A heat map of the ST data for AR for replicate 1, and d, for replicate 2. The immunostained section was cut after replicate 2 and is therefore spatially closest to that section. Accordingly, the coherence between the RNA expression and amount of protein is best for replicate 2.  TRPM8  IFI6  H2AFJ  DHCR24  TRPM8  IFI6  H2AFJ Pre-ADT Post-ADT